Gene involved in growth-promoting function of acetic acid bacteria and uses thereof

ABSTRACT

The present invention provides a gene involved in the growth-promoting function of acetic acid bacteria and a microorganism containing the gene. In particular, the present invention provides a method for enhancing the growth-promoting function of acetic acid bacteria and a method for efficiently producing vinegar containing acetic acid at a high concentration in a short time using such acetic acid bacteria having an enhanced growth-promoting function.

The present application claims priority to, and is the National Phaseunder 35 U.S.C. § 371 of, PCT International Application No.PCT/JP2004/008797, which has an International filing date of Jun 16,2004, which designated the United States of America. This applicationfurther claims priority under 35 U.S.C. § 119 to JP 2003-183047 filed onJun. 26, 2003. The entire contents of each of these applications ishereby incorporated by reference.

TECHNICAL FIELD

The present invention relates to an acetic acid-resistant microorganism,and more specifically, to a gene encoding a protein having agrowth-promoting function derived from a microorganism, a microorganismwherein the number of copies of such gene is amplified, and a method forproducing vinegar using such microorganisms.

BACKGROUND ART

Acetic acid bacteria are microorganisms broadly utilized for vinegarproduction. In particular, acetic acid bacteria belonging to the genusAcetobacter and the same belonging to the genus Gluconacetobacter areused for industrial acetic acid fermentation.

In acetic acid fermentation, ethanol contained in media is oxidized andconverted into acetic acid by acetic acid bacteria. As a result, aceticacid is accumulated in the media. Acetic acid is also inhibitory onacetic acid bacteria. As the amount of accumulated acetic acid increasesand the acetic acid concentration in media becomes higher, the growthability and the fermentation ability of acetic acid bacteria graduallydecrease.

In particular, growth induction period, that is, the period until aceticacid bacteria actually start to grow, and then it becomes possible toconfirm the accumulation of acetic acid, tends to be longer as theacetic acid concentration becomes higher.

Hence, in acetic acid fermentation, it is desired to further shorten thegrowth induction period, even in the case of a higher acetic acidconcentration. As a means for this purpose, a method has been disclosedthat involves adding PQQ(4,5-dihydro-4,5-dioxo-1H-pyrrolo[2,3-f]quinoline-2,7,9-tricarboxylicacid) to a fermention liquid to promote growth, so as to shortenso-called the growth induction period (e.g., see JP Patent Publication(Kokai) No. 61-58584 A (1986)).

However, obtaining PQQ in large quantities is difficult, and PQQ isexpensive. Thus, implementation of such a method at industrial scale hasbeen considered to be uneconomical. Accordingly, efforts have been madeto breed and improve acetic acid bacteria by promoting the growth(resistance to acetic acid) of acetic acid bacteria in the presence of ahigh acetic acid concentration, cloning genes encoding proteins having afunction capable of shortening so-called the growth induction period(genes involved in growth promotion), and using the genes involved ingrowth promotion.

However, no genes involved in growth promotion of acetic acid bacteriahave been isolated so far. Under such circumstances, isolation of anovel gene having a growth-promoting function and encoding a proteinthat has functions to promote at a practical level the growth(resistance to acetic acid) of acetic acid bacteria in the presence of ahigh acetic acid concentration and to shorten the growth inductionperiod, and generating acetic acid bacteria having a stronger growthfunction using the gene involved in growth promotion have been desired.

DISCLOSURE OF THE INVENTION

The objects of the present invention are to isolate a novel gene havinga growth-promoting function and encoding a protein capable of improvingat a practical level a growth function (resistance to acetic acid) inthe presence of a high acetic acid concentration and of shortening aso-called growth induction period, to breed an acetic acid bacteriumhaving a better growth-promoting function using the gene having suchgrowth-promoting function, and to provide a method for efficientlyproducing vinegar with a high acetic acid concentration using the aceticacid bacterium.

We generated a hypothesis that a specific gene encoding a protein havinga growth-promoting function that is absent in other microorganisms couldbe present in acetic acid bacteria that are capable of growing andfermenting even in the presence of acetic acid. We then attempted toisolate such gene and thus have succeeded in isolation of such novelgene. Furthermore, we have obtained findings that the use of such geneencoding a protein having a growth-promoting function enablesimprovement in the growth-promoting function and the resistance toacetic acid of microorganisms, as well as efficient production of novelvinegar containing acetic acid at a high concentration. Thus, we havecompleted the present invention.

The present invention is as described in the following (1) to (8).

-   (1) A protein of the following (A) or (B):    -   (A) a protein containing the amino acid sequence shown in SEQ ID        NO: 2; or    -   (B) a protein containing an amino acid sequence derived from the        amino acid sequence shown in SEQ ID NO: 2 by substitution,        deletion, insertion, addition, or inversion of 1 or several        amino acids and having a growth-promoting function.-   (2) A DNA, which encodes the following protein (A) or (B):    -   (A) a protein containing the amino acid sequence shown in SEQ ID        NO: 2; or    -   (B) a protein containing an amino acid sequence derived from the        amino acid sequence shown in SEQ ID NO: 2 by substitution,        deletion, insertion, addition, or inversion of 1 or several        amino acids and having a growth-promoting function.-   (3) A DNA of the following (A), (B), or (C):    -   (A) a DNA containing the nucleotide sequence of nucleotide Nos.        180 to 1376 in the nucleotide sequence shown in SEQ ID NO: 1;    -   (B) a DNA being capable of hybridizing under stringent        conditions to a DNA consisting of a sequence complementary to        the nucleotide sequence of nucleotide Nos. 180 to 1376 in the        nucleotide sequence shown in SEQ ID NO: 1, and encoding a        protein having a growth-promoting function; or    -   (C) a DNA being capable of hybridizing under stringent        conditions to a DNA consisting of a nucleotide sequence that is        produced from a part of the nucleotide sequence of nucleotide        Nos. 180 to 1376 in the nucleotide sequence shown in SEQ ID NO:        1, having a function as a primer or a probe, and encoding a        protein having a growth-promoting function.-   (4) A recombinant vector, which contains the DNA of (2) or (3)    above.-   (5) A transformant, which is transformed with the recombinant vector    of (4) above.-   (6) A microorganism having an enhanced growth-promoting function,    wherein the number of copies of the DNA of (2) or (3) above is    amplified within a cell.

Examples of the above microorganisms include acetic acid bacteriabelonging to the genus Acetobacter or the genus Gluconacetobacter.

-   (7) A method for producing vinegar, which comprises culturing the    microorganism of (6) above in a medium containing alcohol and    causing the microorganism to generate and accumulate acetic acid in    the medium.-   (8) Vinegar containing acetic acid at a high concentration, which is    obtained by the method of (7) above.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of the restriction enzyme map of a genefragment (containing pS10 and ompA) derived from Gluconacetobacterentanii.

FIG. 2 shows the process of culturing a transformant in a mediumcontaining acetic acid, which has an amplified number of copies of agene that is derived from Gluconacetobacter entanii and has agrowth-promoting function.

FIG. 3 shows the amino acid sequence (SEQ ID NO: 2) of a protein encodedby a gene that is derived from Gluconacetobacter entanii and is involvedin a growth-promoting function.

FIG. 4 shows the construction scheme and the restriction enzyme map forpGI18.

BEST MODE OF CARRYING OUT THE INVENTION

The present invention is described in detail as follows. Thisapplication claims a priority from Japanese patent application No.2003-183047 filed on Jun. 26, 2003, and the content of which describedin the specification and/or drawings is herein incorporated.

1. Isolation of a Gene Encoding a Protein Having a Growth-PromotingFunction

We have developed a method for isolating a gene having agrowth-promoting function from acetic acid bacteria and have attemptedto isolate a gene having such function. According to this isolationmethod, a gene having a growth-promoting function is isolated fromacetic acid bacteria by constructing a chromosomal DNA library of aceticacid bacteria, transforming acetic acid bacteria with the chromosomalDNA library, and then screening for an acetic acid bacterial straincapable of growing within 3 days on agar media in the presence of 1%acetic acid, whereas acetic acid bacteria generally require 4 days togrow on the same media.

By the application of this method to acetic acid bacteria belonging tothe genus Gluconacetobacter, which are actually used for vinegarproduction, we have succeeded for the first time in cloning a novel genehaving a growth-promoting function. The novel gene can improve thegrowth-promoting function by which a growth function (resistance toacetic acid) is enhanced at a practical level in the presence of a highacetic acid concentration and the growth induction period is shortened.

The thus obtained acetic acid resistance gene has homology to someextent with a group of proteins produced by an ompA gene that has beenfound in Escherichia coli, an ompA gene from Caulobacter crescentus, andothers, as a result of a homology search of DDBJ/EMBL/Genbank andSWISS-PROT/PIR. It was presumed that the gene is the ompA gene fromacetic acid bacteria.

Furthermore, the ompA gene of the present invention has 36% homology atthe amino acid sequence level with the ompA gene from Escherichia coliand has 30% homology at the amino acid sequence level with the ompA genefrom Caulobacter crescentus. Because of such extremely low degrees ofhomology, it was confirmed that the ompA gene of the present inventionis somewhat analogous to ompA genes from other microorganisms, but is anovel gene (hereinafter, also referred to as the ompA gene) encoding anovel protein (hereinafter, also referred to as the protein OMPA)specific to acetic acid bacteria.

In the present invention, a transformant having an amplified number ofcopies of the ompA gene was generated by ligating the ompA gene to aplasmid vector and then transforming an acetic acid bacterium with thevector. In such transformant, resistance to acetic acid wassignificantly enhanced (see Example 3). Furthermore, when thetransformant was cultured with aeration in the presence of ethanol, itsability to ferment acetic acid, and particularly, its growth-promotingfunction, were significantly enhanced. Growth-promoting function(resistance to acetic acid) in the presence of a high acetic acidconcentration was also enhanced. Hence, shortened growth inductionperiod, enhanced growth rate, ability to grow in increased acetic acidconcentrations and the like were confirmed (see Examples 2 to 4).Accordingly, it could be confirmed that the ompA gene surely encodes aprotein having a growth-promoting function and that the gene isexpressed so that the function of the protein can be exerted. Hence, wehave expected that vinegar with a high acetic acid concentration can beefficiently produced using a microorganism wherein the number of copiesof the ompA gene is amplified.

2. DNA and Protein of the Present Invention

The DNA of the present invention encodes the ompA gene derived from anacetic acid bacterium and encodes a regulatory sequence of the gene.Furthermore, it is presumed that the DNA encodes a protein having afunction to improve resistance to acetic acid and a growth-promotingfunction (SEQ ID NO: 2).

The DNA of the present invention can be obtained from the chromosomalDNA of Gluconacetobacter entanii as described below.

First, a chromosomal DNA library of Gluconacetobacter entanii, such asthe Acetobacter altoacetigenes MH-24 strain (deposited under accessionnumber FERM BP-491 on Feb. 23, 1984, (original deposition) with theInternational Patent Organism Depositary (Tsukuba Central 6, 1-1-1Higashi Tsukuba, Ibaraki, Japan), the National Institute of AdvancedIndustrial Science and Technology (AIST)), is prepared. The chromosomalDNA can be obtained by a conventional method (e.g., see JP PatentPublication (Kokai) No. 60-9489 A (1985)).

Next, to isolate the ompA gene, a chromosomal DNA library is constructedfrom the above-obtained chromosomal DNA. First, the chromosomal DNA ispartially digested with appropriate restriction enzymes to obtain amixture of various fragments. Through the regulation of time forcleavage reaction and the like so as to regulate the degrees ofcleavage, wide-ranging types of restriction enzymes can be used. Forexample, the chromosomal DNA can be digested by applying Sau3A I to theDNA at a temperature of 30° C. or more, preferably at 37° C., at anenzyme concentration ranging from 1 to 10 units/ml for various reactiontime (1 minute to 2 hours).

Next, the thus cleaved chromosomal DNA fragments are ligated to a vectorDNA that is autonomously replicable within acetic acid bacteria, therebyconstructing a recombinant vectors. Specifically, the vector DNA isreacted with a restriction enzyme (e.g., BamH I, which causes thegeneration of a terminal nucleotide sequence complementary to therestriction enzyme Sau3A I used for the cleavage of the chromosomal DNA)under conditions of a temperature of 30° C. and an enzyme concentrationranging from 1 to 100 units/ml for 1 or more hours, thereby completelydigesting and cleaving the vector DNA.

Next, the mixture of chromosomal DNA fragments obtained as describedabove is mixed with the cleaved vector DNA, and then T₄ DNA ligase isadded and reacted under conditions in which temperature ranges from 4°C. to 16° C. and enzyme concentration ranges from 1 to 100 units/ml for1 or more hours (preferably 6 to 24 hours), thereby obtaining arecombinant vector.

Methods for constructing a chromosomal DNA library from chromosomal DNAare known in the art (e.g., the shot gun method), and are not limited tothe above method.

An acetic acid bacterium that generally requires 4 days to grow in thepresence of 1% acetic acid concentration on an agar medium, such as theAcetobacter aceti No. 1023 strain (deposited under accession number FERMBP-2287 on Jun. 27, 1983, (original deposition) with the InternationalPatent Organism Depositary (Tsukuba Central 6, 1-1-1 Higashi, Tsukuba,Ibaraki, Japan), the National Institute of Advanced Industrial Scienceand Technology) is transformed using the thus obtained recombinantvector. Subsequently, the strains are spread on a 1% aceticacid-containing agar medium, followed by 3 days of culture. Thegenerated colonies are inoculated and cultured in a liquid medium.Plasmids are collected from the thus obtained bacteria, so that DNAfragments containing the ompA gene can be obtained.

A specific example of the DNA of the present invention is a DNA havingthe nucleotide sequence shown in SEQ ID NO: 1. In such DNA, thenucleotide sequence of nucleotide Nos. 180 to 1376 in SEQ ID NO: 1 is acoding region that encodes the protein shown in SEQ ID NO: 2.

The nucleotide sequence shown in SEQ ID NO: 1 or the amino acid sequenceshown in SEQ ID NO: 2 (corresponding to nucleotide Nos. 180 to 1376 ofSEQ ID NO: 1 in FIG. 3) showed 36% homology at the amino acid sequencelevel with the ompA gene from Escherichia coli and showed 30% homologyat the amino acid sequence level with the ompA gene from Caulobactercrescentus as a result of homology search of DDBJ/EMBL/Genbank andSWISS-PROT/PIR. Thus, it was presumed that the relevant gene encodes aprotein OMPA. However, because both homologies were as low as 40% orless, it was evident that the gene is a novel gene differing from thesegenes.

Regarding the DNA of the present invention, the nucleotide sequence ofthe ompA gene encoded by the DNA has been elucidated here. Thus, the DNAcan be obtained by polymerase chain reaction (PCR reaction) usinggenomic DNA of an acetic acid bacterium, Gluconacetobacter entanii, as atemplate, and oligonucleotides synthesized based on the nucleotidesequence as primers, or by hybridization using an oligonucleotidesynthesized based on the nucleotide sequence as a probe, for example.Such DNA having functions as a primer or a probe and prepared from apartial sequence of the ompA gene is also encompassed in the DNA of thepresent invention. Specifically, DNA conisisting of the sequence shownin SEQ ID NO: 3 or 4 can be used as a primer in the present invention,but the DNA of the present invention is not limited thereto. Here,“having functions as a primer or a probe” means to have the length andnucleotide composition of a nucleotide sequence, which enable use as aprimer or a probe. Design of such DNA capable of functioning as a primeror a probe is well-known by persons skilled in the art.

DNA (oligonucleotide) can be synthesized according to a conventionalmethod using various commercially available DNA synthesizers, forexample. Furthermore, PCR reaction can be carried out according to aconventional method using Taq DNA polymerase (produced by TAKARA BIOINC.), KOD-Plus- (produced by TOYOBO CO., LTD.), and the like using theThermal Cycler Gene Amp PCR system 9700 produced by Applied Biosystems.

Furthermore, the OMPA protein of the present invention is encoded by theabove DNA and specifically contains the amino acid sequence shown in SEQID NO: 2. As long as a protein containing the amino acid sequence shownin SEQ ID NO: 2 retains a growth-promoting function, the amino acidsequence may comprise a mutation such as substitution, deletion,insertion, addition, or inversion in a plurality of, and preferably 1 orseveral, amino acids.

For example, the protein of the present invention also includes aprotein containing an amino acid sequence derived from the amino acidsequence shown in SEQ ID NO: 2 by deletion of 1 to 10 and preferably 1to 5 amino acids, addition of 1 to 10 and preferably 1 to 5 amino acids,or substitution of 1 to 10 and preferably 1 to 5 amino acids with otheramino acids. DNA that encodes such protein containing an amino acidsequence mutated as described above and having a growth-promotingfunction can also be obtained by a site-directed mutagenesis method;specifically, by altering the nucleotide sequence for deletion,substitution, insertion, addition, or inversion of amino acids atspecific sites, for example. Moreover, the DNA altered as describedabove can also be obtained by a conventionally known treatment to causemutation.

Furthermore, a variant of the DNA of the present invention encoding aprotein having a growth-promoting function can also be synthesized bythe site-directed mutagenesis method or the like. In addition, tointroduce mutation into the DNA that is a gene, a known technique suchas the Kunkel method and the gapped duplex method or modified methodsaccording thereto can be employed. For example, mutation is introducedusing a kit for introducing mutation, which uses the site-directedmutagenesis method (e.g., Mutan-K (produced by TAKARA BIO INC.) orMutan-G (produced by TAKARA BIO INC.)) or the like. Furthermore,mutation can be introduced into a gene, or a chimeric gene can also beconstructed by techniques such as error-prone PCR, DNA shuffling, or thelike. The error-prone PCR technique and the DNA shuffling techniques areknown in the technical field. For example, regarding the error-pronePCR, see Chen K, and Arnold F H. 1993, Proc. Natl. Acad. Sci. U.S.A.,90: 5618-5622, and regarding the DNA shuffling technique, see Stemmer W.P. 1994, Nature, 370: 389-391 and Stemmer W. P., 1994, Proc. Natl. Acad.Sci. U.S.A. 91: 10747-10751.

Here, “growth-promoting function” in the present invention indicates afunction to promote the growth of microorganisms in the presence ofacetic acid. More specifically, the term means a rapid growth rate or alarge amount of growth of bacteria in the presence of acetic acid.Furthermore, the term also indicates a high upper limit of acetic acidconcentration, at which growth or acetic acid fermentation is possible.Such “growth-promoting function” can also mean a function to enhanceresistance to acetic acid. Whether or not a gene wherein mutation isintroduced as described above encodes a protein having suchgrowth-promoting function can be confirmed by determining the presenceor the absence of growth in a medium containing acetic acid, as shown inthe examples.

Furthermore, it is generally known that the amino acid sequence of aprotein and the nucleotide sequence encoding the protein differ slightlyamong different species, strains, variants, and varieties. DNAs encodingsubstantially identical proteins can be obtained from all acetic acidbacteria, particularly those of species or strains belonging to thegenus Acetobacter or the genus Gluconacetobacter, as well as variants,and varieties thereof.

Specifically, for example, in the nucleotide sequence shown in SEQ IDNO: 1, a DNA that hybridizes under stringent conditions to a DNAconsisting of a part of a nucleotide sequence complementary to thenucleotide sequence consisting of nucleotide Nos. 180 to 1376 or to aDNA consisting of a nucleotide sequence that can be a probe preparedfrom a part of the DNA of nucleotide Nos. 180 to 1376 and that encodes aprotein having a growth-promoting function can be isolated from aceticacid bacteria belonging to the genus Acetobacter or the genusGluconacetobacter, mutated acetic acid bacteria belonging to the genusAcetobacter or the genus Gluconacetobacter, or naturally mutated strainsor varieties thereof. In this way, a DNA encoding a proteinsubstantially identical to the aforementioned protein, that is, aprotein retaining a growth-promoting function, can also be obtained. Theterm “stringent conditions” used herein means conditions wherebyso-called specific hybrids are formed and non-specific hybrids are notformed. It is difficult to precisely represent such conditions innumerical values. For example, such conditions are conditions whereinnucleic acids sharing high homology, such as 70% or higher homology,hybridize to each other, and nucleic acids sharing homology lower thanthis level do not hybridize to each other. Other such examples includegeneral washing conditions for hybridization, such as conditions whereinwashing is carried out at 60° C. with a salt concentration correspondingto 0.1% SDS in the case of 1×SSC.

3. Acetic Acid-Resistant Microorganism (Microorganism having EnhancedGrowth-Promoting Function) of the Present Invention

The DNA of the present invention encodes a protein OMPA having agrowth-promoting function. By the use of the DNA of the presentinvention, a microorganism having an enhanced growth-promoting functionin the presence of acetic acid, that is, a microorganism having enhancedresistance to acetic acid, can be produced.

The growth-promoting function of a microorganism can be enhanced, forexample, by ligating the ompA gene to a recombinant vector andtransforming a microorganism with the vector so as to amplify theintracellular number of copies of the gene, or by ligating a structuralgene portion of the gene and a promoter sequence that efficientlyfunctions in a microorganism to a recombinant vector and transformingthe microorganism with the vector, so as to amplify the number of copiesof the gene and to enhance the gene expression.

The recombinant vector of the present invention can be obtained byligating the DNA encoding the OMPA protein as described in the abovesection “2. DNA and protein of the present invention” to an appropriatevector. A transformant can be obtained by transforming a host using suchrecombinant vector of the present invention so that the ompA gene can beexpressed.

As a recombinant vector, a phage or a plasmid that is autonomouslyreplicable within hosts can be used. Examples of plasmid DNA includeplasmids derived from Escherichia coli (e.g., pBR322, pBR325, pUC118,pET16b and so on), plasmids derived from Bacillus subtilis (e.g.,pUB110, pTP5 and so on), and plasmids derived from yeast (e.g., YEp13,YCp50 and so on). Examples of phage DNA include λ phages (e.g., λgt10,λZAP and so on). Furthermore, a transformant can also be prepared usingan animal virus vector such as retrovirus or vaccinia virus, an insectvirus vector such as baculovirus, a bacterial artificial chromosome(BAC), yeast artificial chromosome (YAC), or the like.

Furthermore, target DNA can also be introduced into a host using amulti-copy vector, transposon, or the like. In the present invention,such multi-copy vector or transposon is also included in the recombinantvector of the present invention. Such multi-copy vector includes pUF106(e.g., see Fujiwara, M. et al., Cellulose, 1989, 153-158), pMV24 (e.g.,see Fukaya, M. et al., Appl. Environ. Microbiol., 1989, 55: 171-176),pGI18 (e.g., see the specification of JP Patent Application 2003-350265;Example 3), pTA 5001 (A), and pTA 5001 (B) (e.g., see JP PatentPublication (Kokai) No. 60-9488 A (1985)). A chromosome integration-typevector pMVL1 (e.g., see Okumura, H. et al., Agric. Biol. Chem., 1988,52:3125-3129) can also be used. Moreover, examples of such transposoninclude Mu and IS1452.

To insert the DNA of the present invention into a vector, a method thatinvolves cleaving purified DNA with an appropriate restriction enzymeand then inserting the resultant into a restriction enzyme site or amulti-cloning site of appropriate vector DNA so as to ligate it to thevector is employed, for example.

The DNA of the present invention should be incorporated into a vector sothat the functions of a gene encoded by the DNA are exerted. Hence, inaddition to a promoter and the DNA of the present invention, if desireda cis element such as an enhancer, a splicing signal, a polyA additionsignal, a selection marker, a ribosome-binding sequence (SD sequence),or the like can be ligated to a recombinant vector of the presentinvention. Furthermore, examples of such selection marker include adihydrofolate reductase gene, a kanamycin resistance gene, atetracycline resistance gene, an ampicillin resistance gene, and aneomycin resistance gene or the like.

Furthermore, to substitute a promoter sequence of the ompA gene on thechromosomal DNA with another promoter sequence capable of efficientlyfunctioning in acetic acid bacteria belonging to the genus Acetobacteror Gluconacetobacter, a vector for homologous recombination isconstructed and then homologous recombination is occurred in thechromosome of a microorganism using the vector. Examples of suchpromoter sequence include those derived from microorganisms other thanacetic acid bacteria, such as a promoter sequence of an ampicillinresistance gene of Escherichia coli plasmid pBR322 (produced by TAKARABIO INC.), that of a kanamycin resistance gene of a plasmid pHSG298(produced by TAKARA BIO INC.), that of a chloramphenicol resistance geneof a plasmid pHSG396 (produced by TAKARA BIO INC.), and that of aβ-galactosidase gene or the like. Construction of a vector forhomologous recombination is known by persons skilled in the art. Asdescribed above, through arrangement of the endogenous ompA gene in amicroorganism under control of a strong promoter, the number of copiesof the ompA gene is amplified and thus the expression is enhanced.

Microorganisms to be used for transformation are not specificallylimited, as long as they can express introduced DNA. Examples of suchmicroorganisms include bacteria (e.g., Escherichia coli, Bacillussubtilis, and lactic acid bacteria), yeast, and fungi such as thosebelonging to the genus Aspergillus. In the present invention, it ispreferable to use acetic acid bacteria as the microorganisms used hereinbecause of the purpose of enhancing the growth-promoting functionthereof. Among acetic acid bacteria, bacteria belonging to the genusAcetobacter and those belonging to the genus Gluconacetobacter arepreferable.

An example of bacteria belonging to the genus Acetobacter is Acetobacteraceti. Specifically, for example, the Acetobacter aceti No. 1023 strain(FERM BP-2287), the Acetobacter aceti subsp. xylinum IFO3288 strain, andthe Acetobacter aceti IFO3283 strain can be used.

Furthermore, examples of bacteria belonging to the genusGluconacetobacter include the Gluconacetobacter europaeus DSM6160 strainand Gluconacetobacter entanii. Specifically, for example, theAcetobacter altoacetigenes MH-24 strain (FERM BP-491) can be used.

Methods for introduction of a recombinant vector into bacteria includingacetic acid bacteria are not specifically limited, as long as they aresuitable for introducing DNA into bacteria. Examples of such methodinclude a method using a calcium ion (e.g., see Fukaya, M. et al.,Agric. Biol. Chem., 1985, 49: 2091-2097) and an electroporation method(e.g., see Wong, H. et al., Proc. Natl. Acad. Sci. U.S.A., 1990, 87:8130-8134) or the like.

When yeast is used as a host, Saccharomyces cerevisiae andShizosaccharomyces pombe are used, for example. Methods for introductionof a recombinant vector into yeast are not specifically limited, as longas they are suitable for introducing DNA into yeast. Examples of suchmethod include an electroporation method, a spheroplast method, and alithium acetate method.

Transformants can be selected using the properties of a marker gene on avector to be introduced. For example, when a neomycin resistance gene isused, microorganisms showing resistance to a G418 drug are selected.

In a preferred embodiment of the present invention, a transformant canbe obtained by transferring a recombinant vector containing nucleicacids that have at least the nucleotide sequence shown in SEQ ID NO: 1,for example, a recombinant vector pOMPA1, wherein the nucleic acids havebeen inserted into an acetic acid bacterium-Escherichia coli shuttlevector (multi-copy vector) pUF106, into the Acetobacter aceti No. 1023(FERM BP-2287) strain; or by introducing a recombinant vector pOMPA2,wherein the nucleic acids have been inserted into an acetic acidbacterium-Escherichia coli shuttle vector (multi-copy vector) pGI18,into the Acetobacter aceti subsp. xylinum IFO 3288 strain.

When a growth-promoting function is enhanced as described above inacetic acid bacteria belonging to the genus Acetobacter or the genusGluconacetobacter having ability to oxidize alcohol, the productionamount and production efficiency of acetic acid can be increased.

4. Vinegar Production Method

Microorganisms (acetic acid bacteria) having a selectively enhancedgrowth-promoting function (as a result of the amplification of thenumber of copies of a gene having such growth-promoting function) andhaving ability to oxidize alcohol are produced as described in the abovesection “3. Acetic acid-resistant microorganism of the presentinvention.” Such microorganisms can be used for producing vinegar,because they can grow in the presence of acetic acid and produce aceticacid. Hence, microorganisms having amplified copy number of the ompAgene are cultured in a medium containing alcohol and then caused toproduce and accumulate acetic acid in the medium, so that vinegarcontaining acetic acid at a high concentration can be efficientlyproduced.

Acetic acid fermentation in the production method of the presentinvention may be carried out in a manner similar to a vinegar productionmethod involving a conventional fermentation method using acetic acidbacteria, but a method for fermentation is not specifically limitedthereto. A medium to be used for acetic acid fermentation may be eithera synthetic or a natural medium as long as it contains a carbon source,a nitrogen source, an inorganic substance, and ethanol, and, ifnecessary, contains appropriate amounts of nutrition sources requiredfor the growth of the employed microbial strain.

Examples of a carbon source include various carbohydrates such asglucose and sucrose and various organic acids. As a nitrogen source, anatural nitrogen source such as peptone or lysate of the fermentationmicroorganisms can be used.

Furthermore, culture is carried out under aerobic conditions such asthose of a static culture method, a shaking culture method, or anaeration and agitation culture method. Culture is carried out generallyat 30° C. The pH for a medium generally ranges from 2.5 to 7 andpreferably ranges from 2.7 to 6.5. The pH can also be adjusted usingvarious acids, various bases, buffers, and the like. Culture isgenerally carried out for 1 to 21 days.

Through culture of microorganisms having an amplified number of copiesof the ompA gene, acetic acid is accumulated at a high concentration ina medium. Furthermore, the growth rate of such microorganisms isimproved, so that the acetic acid production rate becomes improved.

According to the present invention, a growth-promoting function can beconferred to microorganisms so as to enhance the growth thereof.Furthermore, in microorganisms having ability to oxidize alcohol andparticularly in acetic acid bacteria, the growth function (resistance toacetic acid) in the presence of a high acetic acid concentration isenhanced and the growth induction period is significantly shortened.Thus, ability to efficiently accumulate acetic acid at a highconcentration in a medium can be conferred to such microorganisms andsuch bacteria. The thus generated microorganisms (acetic acid bacteria)are useful in production of vinegar containing acetic acid at a highconcentration.

EXAMPLES

The present invention will be further described specifically byreferring to examples. However, the technical scope of the presentinvention is not limited by these examples.

Example 1 Cloning of a Gene Derived from Gluconacetobactor entaniihaving a Growth-Promoting Function and Determination of the NucleotideSequence and the Amino Acid Sequence thereof

(1) Construction of Chromosomal DNA Library

The Acetobacter altoacetigenes MH-24 strain (FERM BP-491), which is astrain of Gluconacetobacter entanii, was cultured in shaking culture at30° C. in a YPG medium (3% glucose, 0.5% yeast extract, and 0.2%polypeptone) supplemented with 6% acetic acid and 4% ethanol. After thecultivation, the culture medium was centrifuged (7,500×g for 10minutes), thereby obtaining bacterial cells. From the thus obtainedbacterial cells, chromosomal DNAs were prepared according to achromosomal DNA preparation method (e.g., see JP Patent Publication(Kokai) No. 60-9489 A (1985)).

The chromosomal DNAs obtained in the above manner were partiallydigested with a restriction enzyme Sau3A I (from TAKARA BIO INC.).Escherichia coli-acetic acid bacterium shuttle vector pUF106 wascompletely digested and cleaved with a restriction enzyme BamH I.Appropriate amounts of these DNAs were mixed in and then ligated using aligation kit (TaKaRa DNA Ligation Kit Ver. 2, from TAKARA BIO INC.),thereby constructing a chromosomal DNA library of Gluconacetobacterentanii.

(2) Cloning of a Gene having a Growth-Promoting Function

The chromosomal DNA library of Gluconacetobacter entanii obtained asdescribed above was transformed into the Acetobacter aceti No. 1023strain (FERM BP-2287) that is known to generally require 4 days to growon an agar medium containing 1% acetic acid. The bacterial cells werethen cultured on a YPG agar medium containing 1% acetic acid and 100μg/ml ampicillin at 30° C. for 3 days. Colonies generated within 3 dayswere inoculated and cultured on a YPG medium containing 100 μg/mlampicillin and then plasmids were collected from the obtained bacterialcells. An approximately 2.3-kbp Sau3A I fragment was cloned as shown inFIG. 1, and the plasmid was named pS10.

As described above, a gene fragment was obtained that has agrowth-promoting function enabling the Acetobacter aceti No. 1023 strainto grow within 3 days on an agar medium containing 1% acetic acid,although the strain generally requires 4 days to grow on such an agarmedium containing 1% acetic acid.

(3) Determination of the Nucleotide Sequence of the Cloned DNA Fragment

The above cloned Sau3A I fragment was inserted into the BamH I site ofpUC19, and then the nucleotide sequence of the fragment was determinedby Sanger's dideoxy chain termination method. As a result, thenucleotide sequence shown in SEQ ID NO: 1 was determined. Sequencing wascarried out for the entire region of both DNA strands with all thecleavage points overlapping each other. The thus obtained gene was namedompA.

In the nucleotide sequence shown in SEQ ID NO: 1, the presence of anopen reading frame encoding 399 amino acids shown in SEQ ID NO: 2 (FIG.3) ranging from nucleotide Nos. 180 to 1376 was confirmed.

Example 2 Effect of Shortening the Growth Induction Period in aTransformant with a Gene having a Growth-Promoting Function fromGluconacetobacter entanii

(1) Transformation into Acetobacter aceti

The above ompA gene cloned according to Example 1 from the Acetobacteraltoacetigenes MH-24 strain (FERM BP-491) was amplified by the PCRmethod using KOD-Plus- (from TOYOBO CO., LTD.). The thus amplified DNAfragment was inserted into the restriction enzyme Sma I cleavage site ofthe acetic acid bacterium—Escherichia coli shuttle vector pUF106 (e.g.,see Fujiwara, M. et al., CELLULOSE, 1989, 153-158), so as to prepare aplasmid pOMPA1. The amplified fragment inserted in pOMPA1 isschematically shown in FIG. 1. FIG. 1 shows the restriction enzyme mapof the Gluconacetobacter entanii-derived gene fragment (pS10) clonedusing Sau3A I, the position of the gene having the growth-promotingfunction, and the fragment inserted into pOMPA1.

The PCR method was carried out as described in detail below.Specifically, PCR was carried out under the following PCR conditionsusing a genomic DNA of the Acetobacter altoacetigenes MH-24 strain as atemplate, primer 1 (5′-GTTTCCCGGAATTCCCGTTTCAGCTCCTTC-3′: SEQ ID NO: 3),primer 2 (5′-ATATCTTTCAGGGCATTTGGAGGTATTCCG-3′: SEQ ID NO: 4), andKOD-Plus- (from TOYOBO CO., LTD.).

Specifically, the PCR method was carried out for 30 cycles, eachcomprising 94° C. for 15 seconds, 60° C. for 30 seconds, and 68° C. for1 minute.

The pOMPA1 was transformed into the Acetobacter aceti No. 1023 strain byan electroporation method (e.g., see Wong, H C. et al., Proc. Natl.Acad. Sci. U.S.A., 1990, 87: 8130-8134). The transformant was selectedusing a YPG agar medium supplemented with 100 μg/ml ampicillin and 1%acetic acid.

Plasmids were extracted from the ampicillin-resistant transformant thathad grown on the selection medium within 3 days and then analyzedaccording to a conventional method. Thus, it was confirmed that thestrain retained plasmids having the gene with the growth-promotingfunction.

(2) Acetic Acid Fermentation Test using the Transformant

The ampicillin-resistant transformant obtained as described above havingthe plasmid pOMPA1 and the original Acetobacter aceti No. 1023 strainhaving only the shuttle vector pUF106 were compared in terms of aceticacid fermentation ability.

Specifically, aeration and agitation culture was carried out in a 2.5 LYPG medium containing 1% acetic acid, 4% ethanol, and 100 μg/mlampicillin at 30° C., 400 rpm, and 0.20 vvm using a 5 L mini-jarfermentor (from Mitsuwa Scientific Corp.; KMJ-5A). The strains wereallowed to ferment to an acetic acid concentration of 3%. Subsequently,some of the culture medium was removed, with 700 mL of the culturemedium left in the mini-jar fermentor. A 1.8 L YPG medium containingacetic acid, ethanol, and 100 μg/ml ampicillin was added to theremaining 700 ml of the culture medium to an acetic acid concentrationof 3% and an ethanol concentration of 4%. Acetic acid fermentation wasinitiated again. Aeration and agitation culture was continued whileadding ethanol so as to maintain the ethanol concentration of 1% in themedium. The transformant was compared with the original strain in termsof acetic acid fermentation ability. The results are summarized in Table1.

TABLE 1 Final acetic Growth acid Specific Production inductionconcentration growth rate rate period achieved (%) (OD660/hr) (%/hr)(hr) Original 9.9 0.0162 0.071 54.4 strain Transformant 9.8 0.0213 0.0725.0

Based on the results in Table 1, it can be confirmed that in the case ofthe transformant, the specific growth rate was significantly higher, thegrowth induction period was significantly shortened, and thetransformant was able to efficiently conduct acetic acid fermentation.

Example 3 Enhancement of Acetic Acid Resistance of the Transformant witha Gene having a Growth-Promoting Function from Gluconacetobacter entanii

(1) Construction of Acetic Acid Bacterium-Escherichia Coli ShuttleVector pGI18

pGI18 was constructed using an approximately 3.1-kb plasmid pGI1 derivedfrom the Acetobacter altoacetigenes MH-24 strain (FERM BP-491) andpUC18.

Specifically, bacterial cells were collected from the culture medium ofthe Acetobacter altoacetigenes MH-24 strain (FERM BP-491), lysed usingsodium hydroxide or sodium dodecyl sulfate, treated with phenol, andthen treated with ethanol, thereby purifying plasmid DNA.

The thus obtained plasmid was a circular double-stranded DNA plasmidhaving 3 recognition sites for Hinc II and 1 recognition site for Sfi I.The entire length of the plasmid was approximately 3100 base pairs (bp).Furthermore, the plasmid did not have recognition sites for EcoR I, SacI, Kpn I, Sma I, BamH I, Xba I, Sal I, Pst I, Sph I, or Hind III. Theplasmid was named pGI1 and used for the construction of the vectorpGI18.

The above-obtained plasmid pGI1 was amplified by the PCR method usingKOD-Plus- (from TOYOBO CO., LTD.). The amplification products werecleaved with Aat II. The fragment was inserted into the restrictionenzyme Aat II cleavage site of pUC18, thereby preparing a plasmid pGI18(FIG. 4).

The PCR method was carried out as described in detail below.Specifically, PCR was carried out under the following PCR conditionsusing a plasmid pGI1 as a template and primer A (SEQ ID NO: 6) andprimer B (SEQ ID NO: 7), which have restriction enzyme Aat IIrecognition sites, as primers.

Specifically, the PCR method was carried out for 30 cycles, eachcomprising 94° C. for 30 seconds, 60° C. for 30 seconds, and 68° C. for3 minutes.

As shown in FIG. 4, the thus obtained plasmid pGI18 contained both pUC18and pGI1, and the entire length thereof was approximately 5800 basepairs (5.8 kbp).

The nucleotide sequence of the plasmid pGI18 is shown in SEQ ID NO: 5.

(2) Transformation into Acetobacter aceti subsp. xylinum

The gene derived from the Acetobacter altoacetigenes MH-24 strain (FERMBP-491) having the growth-promoting function obtained in Example 1 wasamplified by the PCR method using KOD-Plus- (from TOYOBO CO., LTD.). Theacetic acid bacterium-Escherichia coli shuttle vector pGI18 constructedin (1) was cleaved with a restriction enzyme Sma I. The amplified DNAfragment was then inserted into the restriction enzyme Sma I cleavagesite of the shuttle vector, so as to prepare a plasmid pOMPA2. Theamplified fragment inserted in pOMPA2 is schematically shown in FIG. 1.FIG. 1 shows the restriction enzyme map of the Gluconacetobacterentanii-derived gene fragment (pS10) cloned using Sau3A I, the positionof the gene having the growth-promoting function, and the fragmentinserted into pOMPA2.

The PCR method was carried out as described in detail below.Specifically, PCR was carried out under the following PCR conditionsusing a genomic DNA of the Acetobacter altoacetigenes MH-24 strain as atemplate, primer 1 (5′-GTTTCCCGGAATTCCCGTTTCAGCTCCTTC-3′: SEQ ID NO: 3),primer 2 (5′-ATATCTTTCAGGGCATTTGGAGGTATTCCG-3′: SEQ ID NO: 4), and usingKOD-Plus- (from TOYOBO CO., LTD.).

Specifically, the PCR method was carried out for 30 cycles, eachcomprising 94° C. for 15 seconds, 60° C. for 30 seconds, and 68° C. for1 minute.

The pOMPA2 was transformed into the Acetobacter aceti subsp. xylinumIFO3288 strain, which is a strain of Acetobacter aceti subsp. Xylinum,by the electroporation method (e.g., see Wong, H C. et al., Proc. Natl.Acad. Sci. U.S.A., 1990, 87:8130-8134). The transformant was selectedusing a YPG agar medium supplemented with 100 μg/ml ampicillin and 1%acetic acid.

Plasmids were extracted from the ampicillin-resistant transformant thathad grown on the selection medium and then analyzed according to aconventional method. Thus, the retention of plasmids having the aceticacid resistance gene was confirmed.

(3) Resistance of the Transformant to Acetic Acid

The ampicillin-resistant transformant having the plasmid pOMPA2 obtainedin (2) above was compared with the original Acetobacter aceti subsp.xylinum IFO3288 strain having only the shuttle vector pGI18 introducedtherein in terms of growth in a YPG medium supplemented with aceticacid.

Specifically, shaking culture (150 rpm) was carried out at 30° C. in 100ml of a YPG medium containing 3% acetic acid and 100 μg/ml ampicillin.The growth of the transformant and that of the original strain in themedium supplemented with acetic acid were compared by measuring bacteriaconcentrations at 660 nm.

As a result, as shown in FIG. 2, in the medium supplemented with 3%acetic acid, it could be confirmed that whereas the transformant(indicated with open circles) could grow, the original Acetobacter acetisubsp. xylinum IFO3288 strain (indicated with closed circles) was unableto grow. Thus, the function to enhance resistance to acetic acid of thegene having the growth-promoting function could be confirmed.

Example 4 Acetic Acid Fermentation Test for the Transformant with a Genehaving a Growth-Promoting Function from Gluconacetobacter entanii

The ampicillin-resistant transformant having the plasmid pOMPA2 obtainedin Example 3 was compared with the original Acetobacter aceti subsp.xylinum IFO3288 strain having only the shuttle vector pGI18 introducedtherein in terms of ability to ferment acetic acid.

Specifically, a 5 L mini-jar fermentor (from Mitsuwa Scientific Corp.;KMJ-5A) was filled with a YPG medium having an acetic acid concentrationof 1% and an alcohol concentration of 4%. The transformant or theoriginal strain was inoculated at 0.4% onto the medium. Aeration andagitation culture was then initiated at a fermentation temperature of32° C., 500 rpm, and 0.20 vvm. When the acetic acid concentrationincreased to 4% as fermentation proceeded, addition of a raw-materialmedium (alcohol concentration of 7.8% and acetic acid concentration of0.26%), which had been prepared by mixing a 17.9% solution ofsaccharified rice, a 3.2% acetic acid fermentation solution, 7.8%alcohol, and 71.1% water, was initiated. Fermentation was furthercontinued until the acetic acid concentration was increased to 7.2%.

When acetic acid concentration was increased to 7.2%, continuousfermentation was carried out while adjusting the addition rate of theraw-material medium so as to be able to maintain the acetic acidconcentration.

In terms of the addition rate of the raw-material medium, that is,addition rate (proportional to production rate), the transformant andthe original strain were compared. The results are shown in Table 2.

Moreover, the transformant and the original strain were also compared interms of acetic acid fermentation ability, when the addition rate in thecase of the transformant was adjusted to be almost equivalent to that ofthe original strain at the time of continuous fermentation at an aceticacid concentration of 7.2%. The results are shown in Table 3.

TABLE 2 Acetic acid Bacteria concentration concentration Addition rate(%) (OD660) (g/hr) Original strain 7.17 0.675 87.2 Transformant 7.230.675 98.5

TABLE 3 Acetic acid Bacteria concentration concentration Addition rate(%) (OD660) (g/hr) Original strain 7.24 0.712 87.1 Transformant 7.640.695 91.1

Based on the results in Table 2, it was shown that also in the case ofcontinuous acetic acid fermentation, productivity (addition rate of theraw-material medium) was higher and better in the case of thetransformant compared with the original strain.

Furthermore, based on the results in Table 3, it was revealed that whencontinuous acetic acid fermentation was carried out at constantproductivity (addition rate of the raw-material medium), thetransformant could perform continuous acetic acid fermentation with ahigher acetic acid concentration and had better resistance to aceticacid compared with the original strain.

INDUSTRIAL APPLICABILITY

The present invention provides a novel gene having a growth-promotingfunction. A strain that can be obtained through the use of the gene hasan enhanced growth function (resistance to acetic acid) in the presenceof a high acetic acid concentration, a shortened growth inductionperiod, and improved resistance to acetic acid. Such strain can be usedfor highly efficient production of vinegar with a high acetic acidconcentration. Hence, the present invention is useful in the highlyefficient production of vinegar with a high acetic acid concentration.

All publications, patents, and patent applications cited herein areincorporated herein by reference in their entirety.

Sequence Free Text

SEQ ID NOS: 3, 4, 6, and 7: synthetic oligonucleotides

1. An isolated DNA, which encodes a protein comprising the amino acidsequence according to SEQ ID NO:2.
 2. An isolated DNA comprising thenucleotide sequence of nucleotide Nos. 180 to 1376 in the nucleotidesequence shown in SEQ ID NO:
 1. 3. A recombinant vector, which comprisesthe DNA according to claim 1 or
 2. 4. A transformant, which is an aceticacid bacterium belonging to the genus Acetobacter or the genusGluconacetobacter transformed with the recombinant vector according toclaim
 3. 5. A method for producing vinegar, which comprises culturingthe transformant according to claim 4 in a medium containing alcohol andcausing the transformant to generate and accumulate acetic acid in themedium.